The role of the insulin receptor C-terminal domain in the regulation of insulin signal transduction was explored with a variety of synthetic peptides. One of the peptides, termed peptide HC whose structure corresponds to an unique region of the insulin proreceptor sequence, enhanced insulin-stimulated autophosphorylation of the insulin receptor in cell-free systems and in semi-permeabilized cells at concentrations where there were no detectable effect on basal autophosphorylation levels or on receptor dephosphorylation. A lipophilic analogue of peptide HC, stearyl-peptide HC, added to intact Chinese hamster ovary cells transfected with an expression plasmid encoding the human insulin receptor (CHO/HIRc) enhanced significantly insulin-stimulated insulin receptor autophosphorylation while having no effect on ligand-stimulated receptor phosphorylation activity in CHO cells overexpressing either the IGF-1 receptor or the EGF receptor. Addition of stearyl-peptide HC to CHO/HIRc cells resulted in a significant increase in phosphatidylinositol 3'-kinase and mitogen-activated protein kinase activities in response to insulin. Finally, it was found that peptide HC specifically bind to the insulin receptor BETA-subunit but not with the EGF receptor. Taken together our data demonstrate that a pentadecapeptide related to the C-terminus of the insulin receptor bind to the receptor beta-subunit and that this interaction may contribute to the increased receptor~s intrinsic activity and signal transduction.